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Biomedical subjects

D M Chuang

Publications and source records attributed to D M Chuang.

At least 91 records · Page 5Linked to original sources
PubMed1991

Endothelin-induced activation of phosphoinositide turnover, calcium mobilization, and transmitter release in cultured neurons and neurally related cell types.

Endothelin (ET)-related peptides robustly stimulated [3H]-inositol phosphate (IP) formation in cultured cerebellar granule cells, astrocytes, and C6 glioma cells. Their agonist selectivities were ET-1 = ET-2 greater than or equal to sarafotoxin S6b greater than ET-3 greater than big ET-1 for granule cells and ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for cerebellar astrocytes and C6 glioma cells. These effects were Ca(2+)-dependent but insensitive to antagonists of L-type Ca2+ channels and the Na+/Ca2+ antiporter. Pretreatment of cells with ET-1 or S6b induced homologous desensitization of phosphoinositide (PI) response mediated by ET receptors. Long-term pertussis toxin (PTX) treatment attenuated the phosphoinositide (PI) response in astrocytes and glioma but not in granule cells. ET-1 and its related peptides increased [Ca2+]i in C6 glioma by two distinct pathways: IP3-induced Ca2+ mobilization or receptor-operated Ca2+ influx. La3+, Mn2+, and Cd2+ inhibited the Ca2+ influx and sustained PI turnover, while Ca2+ mobilization was attenuated by phorbol ester and TMB-8. ET-induced Ca2+ influx was essential for the sustained [Ca2+]i increase and PI turnover. Homologous desensitization of [Ca2+]i increase was also noted. In cerebellar granule cells, ET evoked the release of [3H]D-aspartate from these neurons. This action appears to be dependent on PI hydrolysis and [Ca2+]i increase and modulated by protein kinase C.

Animals↗
PubMed1990

Differential down-regulation of beta 1- and beta 2-adrenergic receptor mRNA in C6 glioma cells.

C6 glioma cells possess both beta 1- and beta 2-adrenergic receptors. In response to exposure to isoproterenol, these cells down-regulated the mRNA for both beta 1- and beta 2-adrenergic receptors in a manner that indicates an independent regulatory mechanism for each subtype. In particular, the mRNA species for the beta 1-adrenergic receptor initially increased two-fold during the first hour of exposure before decreasing to 40% of initial levels at 4 hours after exposure. In contrast, the beta 2 mRNA species decreased rapidly and monotonically to 20% of initial levels by 2 hours. The unique response to isoproterenol of each subtype was blocked by the appropriate subtype-specific antagonists, betaxolol and ICI 118,551. In addition, beta-adrenergic receptor mRNA down-regulation was observed in association with contact inhibition, suggesting that events other than receptor occupancy can regulate beta-adrenergic receptor mRNA levels.

Adrenergic beta-Antagonists↗
PubMed1990

Comparative studies of phosphoinositide hydrolysis induced by endothelin-related peptides in cultured cerebellar astrocytes, C6-glioma and cerebellar granule cells.

Effects of endothelin (ET) homologues (ET-1, 2, 3 and sarafotoxin S6b) and its precursor (big ET-1) on phosphoinositide (PI) turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-1 = ET-2 greater than or equal to S6b greater than ET-3 greater than big ET-1 for granule cell neurons, while it was ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for astrocytes and C6-glioma cells. Short-term pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-1-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and C6-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C6-gliomas, but not in granule cells. Thus, phospholipase C-coupled ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis.

Animals↗
PubMed1990

Endothelin-1 stimulates the release of preloaded [3H]D-aspartate from cultured cerebellar granule cells.

We have recently reported that endothelin-1 (ET) induces phosphoinositide hydrolysis in primary cultures of rat cerebellar granule cells. Here we found that ET in a dose-dependent manner (1-30 nM) stimulated the release of preloaded [3H]D-aspartate from granule cells. The ET-induced aspartate release was completely blocked in the absence of extracellular Ca2+, but was unaffected by 1 mM Co2+ or 1 microM dihydropyridine derivatives (nisoldipine and nimodipine). At higher concentration (10 microM) of nisoldipine and nimodipine, the release was partially inhibited. Short-term pretreatment of cells with phorbol 12,13-dibutyrate (PDBu) potentiated the ET-induced aspartate release, while long-term pretreatment with PDBu attenuated the release. Long-term exposure of cells to pertussis toxin (PTX), on the other hand, potentiated the ET-induced effects. Our results suggest that ET has a neuromodulatory function in the central nervous system.

Animals↗
PubMed1990

Characterization of two distinct 5-HT receptors coupled to adenylate cyclase activation and ion current generation in NCB-20 cells.

The level of cyclic AMP in NCB-20 cells was increased by serotonin (5-HT), 5-methoxytryptamine and 2-methyl-5-HT with EC50 of 0.5 +/- 0.1, 1.0 +/- 0.1, 10 +/- 0.1 microM, respectively. The 5-HT-mediated increase of cyclic AMP content was completely blocked by metergoline but unaffected by 5-HT3 antagonists, ICS 205-930, MDL 72222, quipazine and 5-HT2 antagonist, ketanserin. Putative 5-HT1A agonists (8-OH-DPAT, ipsapirone, and buspirone) and 5-HT1B agonists (TFMPP and m-CPP) affected neither basal nor forskolin-dependent cyclic AMP accumulation. Receptor binding studies suggest that NCB-20 cells are devoid of 5-HT1A and 5-HT1B receptor sites. Application of 5-HT onto NCB-20 cells resulted in membrane depolarization by an evoked inward current which displayed rapid desensitization. 5-HT-mediated current had a reversal potential around 0 mV and was potently and reversibly inhibited by ICS 205-930. Our data suggest that in NCB-20 cells the 5-HT3 receptor is involved in the generation of inward currents, while the 5-HT receptor coupled to adenylate cyclase does not seem to correspond to any of the known receptor subtypes.

5-Methoxytryptamine↗
PubMed1990

Regulation by batrachotoxin, veratridine, and monensin of basal and carbachol-induced phosphoinositide hydrolysis in neurohybrid NCB-20 cells.

Batrachotoxin (BTX), veratridine and monensin induced a time- and dose-dependent increase of [3H]-inositol monophosphate (3H-IP1) accumulation in the presence of lithium in prelabeled neurohybrid NCB-20 cells. A decrease of NaCl concentration to less than 30 mM markedly increased basal 3H-IP1 accumulation; however, the percentage of stimulation induced by these three agents remained unchanged even in the complete absence of sodium. The stimulation of phosphoinositide hydrolysis induced by these agents was detected in the absence of lithium but was largely prevented in the calcium-free medium. Tetradotoxin (TTX) blocked effects of BTX and veratridine (IC50 approximately 20nM), but not that stimulated by monensin. Thus, calcium-dependent activation of phospholipase C by these agents did not involve the entry of sodium or lithium. BTX and monensin also induced greater than additive effects on carbachol-induced 3H-IP1 accumulation. These effects were also TTX-sensitive and involved an increase in the Vmax and a decrease in the EC50 for carbachol. Veratridine provoked strikingly different effects on carbachol-dependent phosphoinositide turnover, depending on the passage number of the cells.

Batrachotoxins↗
PubMed1990

Expression of neurotrophic activity in Xenopus oocytes injected with mRNA from wounded rat cerebral cortex.

Injury to the cerebral cortex of the rat brain has been shown to induce the expression of neurotrophic factors for dissociated peripheral and central neurons in culture. We confirm this phenomenon and report that Xenopus laevis oocytes injected with mRNA extracted from wounded rat cortex expressed similar neurotrophic activity. To detect the low amounts of neurotrophic factors that could be expected from the oocyte translation system, a miniaturization of the assay for neurotrophic and cell-surviving activity was developed using Terasaki microtiter plates for culture of chicken embryo sympathetic ganglion cells. Messenger RNA (mRNA) was size-fractionated on a sucrose gradient and RNAs from each fraction were injected into oocytes. Neurotrophic activity was recovered from the homogenates and from the incubation media of oocytes injected with mRNA from 7 day post-lesion cortex. Messenger RNAs in the active fractions ranged in size from 0.8 to 1.8 kb. As much as 20% of the activity was secreted by the oocytes. No significant neurotrophic activity was detected from oocytes injected with mRNA fractions extracted from the cortex of control rats or from other gradient fractions from post-lesion cortex.

Animals↗
PubMed1990

Maitotoxin induces phosphoinositide turnover and modulates glutamatergic and muscarinic cholinergic receptor function in cultured cerebellar neurons.

Maitotoxin (MTX) stimulated inositol phosphate (IP) formation in primary cultures of rat cerebellar granule cells. MTX-induced IP production was dependent on extracellular Ca2+ but independent of extracellular Na+. The stimulation of IP formation elicited by MTX was unaffected by pretreatment of cells with phorbol dibutyrate, pertussis toxin, and a variety of Ca2+ entry blockers, such as nimodipine, nisoldipine, Co2+, and Mn2+. The presence of MTX markedly attenuated IP production induced by carbachol and glutamate, with no apparent effect on the responses to norepinephrine (NE), histamine, 5-hydroxytryptamine (5-HT), and endothelin-1. The inhibition of the carbachol- and glutamate-induced responses by MTX was dose dependent with IC50 values of 1.2 and 0.5 ng/ml, respectively. Pretreatment of cells with a lower concentration of MTX (0.3 ng/ml) also attenuated carbachol- and glutamate-induced IP formation, in a time-dependent manner, with a decrease observed after 30 min prestimulation, but failed to affect NE-, histamine-, 5-HT-, endothelin-1, and sarafotoxin S6b-induced responses. Thus, MTX elicited a marked Ca2(+)-dependent phosphoinositide (PI) turnover in cerebellar granule cells and selectively inhibited carbachol- and glutamate-induced PI hydrolysis. Possible mechanisms underlying these selective modulations are discussed.

Animals↗
PubMed1989

Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells.

The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. This effect was prevented by 5-HT2 specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT2 receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.

Animals↗
PubMed1989

Cross-linking of [125I]beta-endorphin to mu-opioid receptors during development.

Radioiodinated human beta-endorphin was cross-linked to opioid receptors from rat brain membranes using the bifunctional reagents bis-[2-(succinimidooxycarbonyloxy)ethyl] sulfone (BSCOES) and disuccinimidyl suberate (DSS). Major radiolabeled bands migrated with Mr values of 65,000, 55,000 and 33,000, however the presence of the 55 kDa band was variable. The 65 kDa band was characterized as the mu-receptor: the binding of [125I]beta-endorphin to this band was displaced by mu-selective ligands and blocked by alkylation of the receptor by mu-specific, but not delta-specific alkylating agents. The cross-linked receptor underwent alterations in mol. wt. during development. Early in development, embryonic day 18 and postnatal day 1, the [125I]beta-endorphin-labeled material migrated as a single band of mol. wt. 55 kDa. By day 21 postnatally the higher mol. wt. band of 65 kDa was present, as was material of 53, 47 and 43 kDa. Although the protein labeled early in development migrated with a mol. wt. of 55 kDa similar to the delta-receptor isolated from NG108-15 neuroblastoma-glioma cells, competition studies suggest this protein is not the delta-receptor. The 65 kDa band, tentatively identified as the mu-receptor, was present in adults but not detected in neonates, despite competition binding data indicating the presence of mu-sites. The results suggest that the 55 kDa band found in the 1-day-old neonate may be an immature form of the mu-opioid receptor that undergoes posttranslational modification, perhaps glycosylation, during development.

Alkylation↗
PubMed1989

Rebound increase of basal cAMP level in NG108-15 cells during chronic morphine treatment: effects of naloxone and chloramphenicol.

The effects of morphine on the basal cAMP level in the neuroblastoma X glioma NG108-15 hybrid cell line have been studied. Morphine (10 microM) added to the incubation media at hr 0 caused a rapid and significant decrease in the cAMP level up to hr 1; the level then slowly returned to the control at hr 6, and gradually increased to its peak at hr 36, returning to the control at hr 60. These results provide the first evidence for a delayed rebound increase of cAMP during morphine treatment. Naloxone (10 microM) added at hr 0 concomitantly with morphine blocked the morphine-induced decrease in cAMP level at hr 1 and attenuated its increase at hr 36. However, when naloxone was added at hr 5.5, the cAMP level significantly increased at hr 6, and at hr 36 the cAMP level increase was the same as in the case of morphine alone. Furthermore when naloxone was added 0.5 hr prior to harvesting the cells at hr 6, 12, 24 and 36, the cAMP level showed an immediate increase at each time point up to about the same level as observed with morphine alone at hr 36. Chloramphenicol, a protein synthesis inhibitor (100 microM) itself caused little or no change in the cAMP level. Added 30 min before morphine, chloramphenicol decreased the morphine-induced rebound increase at hr 36 in a concentration-dependent manner without any significant effect on cAMP decrease at hr 1. However when chloramphenicol was added at hr 5.5, the morphine-induced rebound increase at hr 36 was also attenuated, thereby suggesting an involvement of macromolecular synthesis in the rebound increase of cAMP which may be used as a model for the development of morphine dependence.

Chloramphenicol↗
PubMed1989

Homologous desensitization of muscarinic cholinergic, histaminergic, adrenergic, and serotonergic receptors coupled to phospholipase C in cerebellar granule cells.

Cultured cerebellar granule cells express phospholipase C-coupled muscarinic cholinergic, histaminergic, alpha 1-adrenergic, and serotonergic receptors. In an attempt to study desensitization of these neurotransmitter receptors, cells were prestimulated with saturating concentrations of carbachol, histamine, norepinephrine, or serotonin during the labeling of cells with myo-[3H]inositol and then rechallenged with various receptor agonists for their ability to elicit accumulation of [3H]inositol monophosphate in the presence of lithium. Prestimulation with each of these receptor agonists was found to cause a time-dependent desensitization to subsequent stimulation with the desensitizing agonist. Thus, prestimulation for 0.5, 4, and 18 h decreased carbachol response to 87 +/- 4, 52 +/- 2, and 40 +/- 1% of the control, respectively; histamine response to 37 +/- 2, 24 +/- 2, and 18 +/- 2%, respectively; norepinephrine response to 55 +/- 5, 14 +/- 1, and 10 +/- 1%, respectively; and serotonin response to 36 +/- 1, 18 +/- 1, and 9 +/- 2%, respectively. In all cases, the responses mediated by receptors which were not prestimulated remained virtually unchanged, thus indicating homologous desensitization. Dose-response studies indicate that the desensitization was associated with a major reduction in the maximal extent of agonist-induced responses. The basal accumulation was markedly enhanced following 0.5- and 4-h prestimulation, but returned to near normal after 18-h pretreatment. Biologically active phorbol ester, 4 beta-phorbol 12-myristate 13-acetate, rapidly attenuated basal phospholipase C activity, as well as the responses mediated by carbachol, histamine, norepinephrine, and serotonin, suggesting that activation and translocation of protein kinase C might play a role in the desensitization of phospholipase C-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Substrates for protein kinase C in a cell free preparation of rat aorta smooth muscles.

Protein phosphorylation has been studied in a cell free system of rat aorta smooth muscles. Addition of Ca2+ caused phosphorylation of several proteins. The addition of phosphatidylserine or calmodulin together with Ca2+ further increased the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons. The activators of protein kinase C, 12-0-tetradecanoylphorbol-13-acetate and 1,2-diolein, increased phosphorylation of the protein bands of similar molecular weight to those increased by phosphatidylserine in the presence of Ca2+, whereas the biologically inactive phorbol ester, 4 alpha-phorbol-12,13 didecanoate (4 alpha PDD) failed to change the pattern of protein phosphorylation. These results show that proteins present in smooth muscle of rat aorta with molecular weights of 20 and 92.5 kilodaltons are substrates for protein kinase C.

Animals↗